Quantitative Correlation between the Distribution of Anions and the pH Difference across the Mitochondrial Membrane

The factors regulating the distribution of anions between the inner and outer mitochondrial phase have been investigated in the presence of respiratory inhibitors and oligomycin. As the external pH is decreased, the ratio of the intra‐ to extramitochondrial concentration of added anions increases with acetate, malate and phosphate, and with citrate in the presence of malate. In contrast, the uptake of thiocyanate does not change significantly with the pH. In the pH range used, the log of the anion distribution ratio is proportional to the pH difference across the mitochondrial membrane for acetate, malate and phosphate. The anion distribution ratio is highly sensitive to pH at low external concentration and becomes largely independent of pH at high external concentration. In double reciprocal plots of intra‐ against extramitochondrial concentration, it was found that the capacity ( A m ) for the uptake of phosphate, acetate, malate and succinate is independent of pH. The slopes ( A 1/2 / A m ) increase with pH due to the increase of the external concentration required for half maximal uptake. The distribution ratio of phosphate and malate is proportional to the relative “free capacity” (α) at all the external pH tested. The dependence of the distribution ratio on the external pH is maximal for α= 1 and nil for α= 0. The following relation holds for the dependence of the slope A 1/2 / A m on Δ pH: log A m / A 1/2 = a + b Δ pH. This relation was used to obtain the equation describing the dependence of the anion distribution on the Δ pH and on the capacity. It is concluded that the distribution of anions, such as acetate, phosphate and Krebs cycle intermediates, at equilibrium can be explained essentially as a function of two parameters: (a) the pH difference across the mitochondrial membrane (b) the capacity of the mitochondria for anions.